Great job keep up the good work -UM
weeks 11 &12
week 12 Protein expression protocol for surrogate target
Grow protein for Enzyme Assays on the surrogate target
Docking of NIH_ClinCol3Ded Protein Characterization special protocol
Due to the lack of a good expression in the first trial the expression was redon and special buffers were made to try and make the protein soluble
The band of the protein size was in the nonsoluble fraction due to this it was determined that the protein is insoluble. The change to a surrogate target was made.
week 11 Docking of HF9platesplates
A slight hit was found in this library since the scores were higher than the top ranking ligand in the control docking
Docking of CB_306
The gold results from this docking did not show any good hits. This since the scores were lower than the scores from the control docking Protocol Virtual screen on protein target
purpose is to use GOLD/Hermes to prepare a protein for docking
The homology model that was created in protocol homology model was used to set up for virtual screening on the control ligands.
Control Results from virtual screening control docking
The results of the control docking are shown above this results prove that the control docking on the homology model was a success. The top ranking ligand in this docking was the one found in the protein used for the homology model template. Protocol Ligand Prep
Purpose is to prepare the control ligands for virtual screening
The ligands were concatenated into a single file and taken through a ligand preparation processes using ICM which prepares the ligands by applying the proper protonation states for pH 7, assigning 3-D coordinates and were energy minimized using the MMFF (Merck Molecular Force Field) prior to doing a control docking.
Protocol Protein Characterization
Purpose is to separate protein samples by gel electrophoresis and visualize with a dye to determine purity and yield
The gel did not show a strong band in the correct ranged due to this the protein might be a membrane protein
Protocol Protein Purification
Purpose is to purify the protein using the affinity tag and Ni-NTA resin
target protein was purified via affinity tag and the end product was taken to protein characterization protocol
weeks 9&10
Week 10 Protocol Protein Expression Scaled Up
Purpose is to make protein for enzyme assays
Plate of BL21 cells with inserted vector pNIC-Bsa4 containing the target DNA sequence plates of 10ul
plate of BL21 cells with inserted vector pNIC-Bsa4 containg the target DNA sequence plate with 50uL coulteure
option B for the small culture was chosen on friday night the small culture was left to grow overnight
For this option a small scale growth of bacteria is grown overnight this bacteria will serve as the starting point for the large scale induction step
For the second day the induction step option B was chosen which is let in shaker incubator for 4 hrs at 37oC
Bacteria from the Overnight culture was transferred into pre-warm LB media until a 0.1 absorbance at 600nm was obtained at which the LB with the BL21 bacteria was placed in the shaker incubator until a .5 absorbance was obtained at which the IPTG (500 uM final) was added to induce the bacteria into producing the target protein.
After 4hrs the bacteria was transferred into two 500ml centrifuge containers and spin down at 6000g for 20min. Following this the liquid was discarded and bleached for 20min with 10% bleach. The pellets were resuspended in 12ml of resuspension buffer and they were then transferred into a 15ml conical tube and placed in the -20oC freezer
Protocol Homology Model Purpose is to find a protein with similar properties as the target protein due to the fact that there is no crystal structure available of the target protein
This protocol has been delayed due to ICM not working properly duing the week but the homology protein has been obtained and is 2OZ5.
20Z5 is the Crystal structure of Mycobacterium tuberculosis protein tyrosine phosphatase PtpB in complex with the specific inhibitor OMTS
Week 9 Protocol Ligand Prep Purpose to obtain and prep positive and negative controls for virtual drug screening of target protein
Data table with properties and specific compounds for negative and positive control ligands this were obtained using the specifications for the protein the negative were obtained at random using compounds with similar characteristics as the postivie controls
Transformation of plasmid in BL21(DE3) cells
Purpose is to insert the plasmid pNIC-Bsa4 with the gene of interest into BL21 expression cells
DNA sequencing analysis
Purpose to blast the sequences obtained from DNA sequencing facility against the known gene of interest in order to confirm a positive clone Sample 4 Forward primer analysis
Portion of sample 6 foward in order to compare it with the reverse Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTA 896 |||||||||||||||||||| ||||| || |||||| |||||||| |||||||| Sbjct 927 AAACTGGGTCTGACGGACGCNAAAAA-GAN-AGCTGAN-AAAGCGTANCTGTACTA 979 *positive clone
Great--looks like you've been working a lot in lab. Captions and analysis could be more thorough. -UM weeks 7&8
Week 8
Protocol Submit to DNA Sequencing**
The purpose is to submit the DNA to sequencing facility to see if the transformed gene in DH5alpha with the inserted pNIC is correct
Protocol Midi Prep Kit - HiSpeed (Round 3)
Purpose is to extract DNA from transformed bacterial cells
For this protocol the Midi prep protocol found inside the kit was used to extract the DNA from the transformed bacteria.
nanodrop showing the concentration of pNIC-Bsa4 after midipreped This concentration is excellent to work with for cloning it should make cutting pNIC more pleasing
Protocol Miniprep
Purpose is to extract DNA from transformed bacterial cells from making master plate protocol
All the consentartions after nano drop were in the range of 77-95ng/uL this were acceptable consentrations and I proceeded to submit to sequencing
Protocol pNIC-Bsa4 cloning
The purpose is to Transfer Gene of Interest into a protein expression vector
Making master plate
**Miniprep protocol was done results at Miniprep protocol
e
LB+Kan+5%suc plate with DH5alpha and pNIC-Bsa4 with the gene of interest inserted from the annealing and transformation
Annealing and Transformation
LB+Kan+5%suc plate with DH5alpha and pNIC-Bsa4 with the gene of interest inserted (ratio of pNIC to gene used for this plate was 1microliter:9microliter)
LB+Kan+5%suc plate with DH5alpha and pNIC-Bsa4 with the gene of interest inserted (ratio of pNIC to gene used for this plate was 2microliter:4microliter)
Cohesive End Generation steps
From both the cut pNIC-Bsa4 and the PCRsquared of my gene of interest the cohesive ends were made
Preparation of pNIC-Bsa4 as Accepting Vector(Round2)
NanoDrop number 1 for cut pNIC Bsa4
NanoDrop number 2 for cut pNIC-Bsa4
**excellent results were obtained from cutting pNIC Bsa4 for the second time will proceed to the next step
Preparation of pNIC-Bsa4 as Accepting Vector(Round1)
Results from nano drop 1 of cut pNIC-Bsa4
Results for nano drop number 2 of cut pNIC-Bsa4
1% Agarose gel for the cut pNIC-Bsa4 round 1 the cut was successful but the final concentration was too low
Due to low level of concentration of the cut pNIC-Bsa4 this will not be useful for the next step the procedure must be redone
Week 7 Protocol transformation for pNIC
After making LB medium I grew more DH5alpha with the vector pNIC-Bsa4 inserted on them. This was done to have extra pNIC incase that the cutting of pNIC does not work.
Protocol LB Medium
purpose to make LB medium to grow bacteria in.
made more LB medium to have ready incase that the cutting of pNIC Bsa4 is unsucessful this will serve to grow more bacteria in.
Protocol LB Agar Kan + Suc plates
purpose to make LB + kan + suc plates to used to grow transformed bacteria
The suc is added to test that the Sac B gene was cut out of pNIC properly since there is a gene that makes a toxic with sucrose which kills the bacteria if the gene is still present in the vector
Protocol Midi Prep Kit-HiSpeed (Round 2)
first nano drop for round two of pNIC-Bsa4
second nano drop for round 2 of pNIC **the average of both nano drops is used to determine final concentration
Round two for Midi Prep results for pNIC-Bsa4 the concentration obtained of pNIC in the second round were slightly better than the first round though it will be hard to do a successful cutting of pNIC Bsa4 an attempt will be done and if unsuccessful results more pNIC will be grown and MIDI preped.
I would like to see more formalized captions bro! Good job with the progress overall though! - Michael T.
week 5 & 6 Week 6
Protocol Midi Prep Kit - HiSpeed
Purpose is to extract DNA from transformed bacterial cells
For this protocol the Midi prep protocol found inside the kit was used to extract the DNA from the transformed bacteria.
In the first attempt to do the midiprep the final concentration of pNIC-Bsa4 was extremely low to even attempt the next step the procedure will be redone during week 7.
The results from the first nanodrop of the midiprep
The results of the second nanodrop of the midiprep
Due to the low concentration of pNIC the procedure will be redone during the following week.
Transformation of competent cells for plasmid prep of pNIC-Bsa4(Day2-3)
The purpose was to use the bacteria colonies obtained from day 1 of the procedure to grow them in a flask of LB media to obtain a large amount in order to have a lot of pNIC-Bsa4.
Following the 12-16hr growth period the bacteria was spinned down on the centrifuge. The pellet was collected and the rest was bleached for 20 min before discarding to the drain.
So far this procedure has been repeated two time due to the fact that when the Miniprep was completed a very low concentration was obtained.
PyMol Refresher
Purpose was to examine a three dimensional structure and analyze it using Pymol. This was supposed to help as a refresher for pyMol
Chain A shown as red chain on the left, Chain B is shown as yellow on the right. The substrate NAP is shown as sticks green and the substrate DU is shown as sticks blue.
Chain A shown as red chain on the right, Chain B is shown as yellow on the left. The substrate NAP is shown as sticks green and the substrate DU is shown as sticks blue. Showing the hydrophobic residues as magenta, ionic residues as orange and polar as pink.
Showing the active site of 3CL9 shown in green. MTX is shown as sticks colored dark green. The polar contacts of MTX to the protein are shown in black. NAP is shown in cyanide.
Alignment of 1U72 and 3CL9 using (RMSD: 1.23). 1U72 is shown as a ribbion, color red. 3CL9 is shown as a ribbon colored green. the active site residues are shown as sticks yellow. MTX is shown as sticks. The polar contacts of MTX to the respective protein are shown in black dashes.
Protein BLAST of residues in the active sites of 1U72 and 3CL9
showing 3HBB as ribbon. With Chain A shown as blue. Chain B shown as Orange. Chain c shown as yellow. Chain D shown as dark pink.
MTX shown as sticks red, active site of 1U72 shown as yellow sticks, polar contacts shown as black dotted line, 3HBB as ribbon. With Chain A shown as blue. Chain B shown as Orange. Chain c shown as yellow. Chain D shown as dark pink.
This lab helped in remembering the basics of PyMol. This lab served as a good review for the program. Specially since the lab used many of the learned techniques from past Pymol experiments. A lot can be obtain from reviewing this information and specially once we start with the virtual screening process and we need to use the pymol to obtain a visual of whats happening in the docking.
Forward: This lab will help in the analysis of the gold results once we do the virtual screening.
Week 5 Transformation of competent cells for plasmid prep of pNIC-Bsa4(Day1)
Purpose to transform bacterial plasmid DNA to make more plasmid DNA
In the first day of this process the DH5Alpha was obtained from the -80C fridge and was added to a conical tube then the vector was added and a 30min ice period was waited. The a 42C 45sec heat shock followed. Then a two minute wait on ice and then the SOC media was added. The tube was placed in the shaker incubater for 30min. The last step was to plate the end product and place in the incubator for overnight incubation. The first time doing this procedure the vector was mistakenly not added to the bacteria making it impossible for them to grow in the LB +kan plate due to the fact that the vector contained the Kan resistance gene.
The procedure was done for a second time and the same result happened it was not until the end of the second incubation period that the problem was figured out but already 2 days of work had been lost due to protocol mistake.
On the third try the plates had a great colony growth
The procedure was done correctly and the process was taken into day two for overnight growth in the LB media + kan.
The resulting plate from the overnight growth.
The second plate with the DH5Alpha +kan +LB this plate was where the colony for the overnight cultivation was taken from since the colonies were more clearly seen.
LB Media and Plates Preparationprotocol
Purpose to make LB media and plates to grow DH5Alpha with pNIC-Bsa4 vector.
For the LB media Bacto-tryptone, Bacto-yeast extract, and NaCL were added at the required concentrations specified in the protocol. For the LB Plates the same concentrations were added just that at the end Bacto-agar was added which is what solidifies the mixture in order to be able to plate. In addition at the end before plating Kanamycin was added to avoid other bacteria to grow.
Both the plates and the LB media had to be Autoclaved prior to the use of it in the growth of bacteria. Extreme clean technique was implemented while plating the Plates due to the fact that a completely sterile plate wanted to be obtained in order to have no contamination. The plates were stored in the 4 degree C fridge and the LB media was stored in the LB media fridge.
PCR Clean up
For the PCR squared clean up I followed the protocol given by Sigma PCR cleanup kit(Red box kit)
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The nanodrop results from the first PCR clean up
In the first attempt to do the clean up the final concentration of PCR product was extremely low due to the fact that a user error was made and the PCR reaction was separated into two containers.
The nanodrop for the second PCR squared cleanup
For the second PCR squared clean up the concentration was still way too low for the target concentration. For this PCR Squared clean up it might have been a faulty PCR squared due to the fact that the gel was made but the strength of the bands was not that high.
The nanodrop of the third PCR Squared Cleanup
In the third PCR squared cleanup the concentration was excellent. This should have been due to the fact that PCR squared was doubled and the amount of elution was half. This made it possible to get a great concentration.
PCR2 squared reaction
Purpose to make extra PCR product to be able to pass it through PCR clean up protocol.
The pcr squared results from the second PCR squared that was done. I forgot to take a gel image of the second PCR squared and I forgot to do a gel for the first PCR squared. But the second pcr squared was successful showing all the bands in the correct size. The thirds PCR squared had to be doubled since for the first two the clean up of the PCR squared was unsuccessful.
The gel shows all the lanes with the PCR squared reaction. This lanes show a size of about 1000bp which is about the size of the gene once the tail primers had been added.
Week 3 & 4
De La O - ok, solid work, but upload your original gel images to Wiki. Use more formalized captions. - Dr. B 092713 Week 4
Protocol Primer Dilution VDS
Purpose dilute primers that have been ordered for cloning.
Excel results for the dilution. This is using the dilution excel page.
Protocol PCR PrimerOverlap VDS with Q5 Polymerase
Purpose to synthesize a gene of interest from scratch
#1 primary and secondary PCR gel
In this gel primary PCR failed and as a result the secondary PCR failed. The problem might have been that the incorrect PCR machine settings were used for this PCR.
#2 Primary PCR
Primary PCR was successful since lane 1 had the smear. This time the Q5 Polymerase recommended times were used. The secondary PCR was left running overnight and was checked the morning after.
#2 Secondary PCR using the oligo tails as tails
Secondary PCR was a success the band was in the correct base pairs size. Though this PCR was done using the tails from the oligos mix since the actual tails had not come in by the time I did Secondary PCR. Though from this we can conclude that the Oligos were made correctly since the gene is now at full size.
#3 Secondary PCR using the tails from tail design
Secondary PCR using the tails that were design was successfully done. This can lead us to the conclusion that the tails were designed correctly and we can now proceed to PCR^2
Week 3
PCR Primer Design Tails for pNIC-Bsa4 cloning
Purpose: Design a forward and a reverse primer for PCR amplifying the CDS of your gene of interest sequence and synthesizing Compatible Ends suitable for Ligation Independent Cloning (LIC) for insertion into the pNIC-Bsa4 as the accepting vector for eventual expression.
Forward Primer Tail:
5’ _TAC TTC CAA TCC ATG AAG AAT TGG GTT AAA GTT _ 3’ _ bp GC Content 33.3_% 0 mM Mg2+ Tm 57.1_ oC 1.5 mM Mg2+ Tm _65.1 oC 2 mM Mg2+ Tm _67.4 oC 4 mM Mg2+ Tm 67_ oC 6 mM Mg2+ Tm 67.5_ oC
Reverse Primer Tail:
Reverse Primer: 5’ _ TATCCACCTTTACTGTTAGTACAGGTACGC 3’ _ bp
GC Content _43.3%
0 mM Mg2+ Tm 60.4_ oC 1.5 mM Mg2+ Tm 68_ oC 2 mM Mg2+ Tm _67.1 oC
Second lane shows the Target protein and the tails
Third lane shows the Target protein with the tails
Primer Dilutions for Assembly Step
Goal to make a dilution from the Oligo's of the gene of interest.
The Oligo's stock solution had a 100uM concentration. The final concentartion dilutios were made to have a 1uM of each oligo. Since my gene of intrest had 22 oligos I added 78uL of Autoclaved nanopure water. This dilution will be used during primary PCR.
Restriction Enzyme Digest
Goal to digest pGBR22 plasmid with restriction enzymes and visualize fragments on gel
This restriction used EcoRI, PvuII, and EcoRI + PvuII
PCR protocol for pGBR22 attempts 2-4
Goal was to Amplify Purple Protein coding sequence in the pGBR22 plasmid using Forward and Reverse Primers. During this protocol pGBR22 went through the process of PCR in order to amplify the DNA. The PCR was tested for amplification using Agarose Gel. Though this procedure will need to be redo during week 3 since the Agarose gel proved that only a small fraction of the DNA was actually amplified. To have more conclusive evidence the procedure will be repeated until a conclusive gel is obtained showing that the pGBR22 was amplified.
Conclusions:
Even though that PCR for pGBR22 had to be redone four times. The information learned from this procedure is invaluable and will help during the PCR for the oligo's that were ordered earlier during week 2. This labs helped me understand the critical measures that need to be implemented in order to obtain a good PCR.
Attempt 4
In attempt number 4 the pgbr22 was finally replicated correctly. Both lane 2 and lane 3 showed a band in the correct size. This leads to the conclusion that pgbr22 was finally replicated correctly. Though this image lacks resolution in the original gel the bands were clearly seen.
Attempt 3
The third time trying to replicated pgbr22 was unsuccessful. I believe that the pgbr22 used for this attempt might have been faulty due to incorrect labeling. In the original image from the gel two small bands in the incorrect size were observed. Though that could have just been contamination in the gel. The next lab day will be dedicated to attempt PCR once again.
Attempt 2
The second attempt to do pcr with pgbr22 was another failed attempt. I believe that what might had happened is that the taq was added much earlier than recommended causing the reaction to start earlier before the PCR machine was working. Another attempt must be made the next day I am in lab.
Week 1 & 2
De La O - good job. Take another try at PCR this week. Dr. B 090913
PCR protocol for pGBR22
Goal was to Amplify Purple Protein coding sequence in the pGBR22 plasmid using Forward and Reverse Primers. During this protocol pGBR22 went through the process of PCR in order to amplify the DNA. The PCR was tested for amplification using Agarose Gel. Though this procedure will need to be redo during week 3 since the Agarose gel proved that only a small fraction of the DNA was actually amplified. To have more conclusive evidence the procedure will be repeated until a conclusive gel is obtained showing that the pGBR22 was amplified.
Gel band #2 had the 1000 ladder, band #3 had the sample A, band #4 had the sample B, band #5 had the sample C and band #6 had the sample D
Band #3 and 4 did not show promising results since the DNA amplification was not detected by the UV light image. Band #5 detected a small number of DNA amplified. While band #6 did not show anything at all since it was the control sample with no DNA in it.
Analyzing DNA Sequence
The goal was to determine the DNA sequence found in the plasmid using the DNA sequencing obtain from the DNA sequencing facility. The DNA sequence was also blasted to compare it to a database of human G+T and a second blast was conducted to compare it to a Nucleotide collection (nr/nt). A virual gel ladder was created to determine where would the DNA sequence be found in a gel.
Original DNA Sequence
NNNNNNNNNNNTATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANCNNNNNCNTTCNNTTNTNCNNNTNNNNNNNNNNNNNNNTTNCCGNNAGNTNNAANNGGGGNNNNNNNNNATNNGNTNNNNNNNNNNCCAAANTGNNNGNNNNGNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNGANCNNNNNNNNNNNNANNNNNNNNNNNCNNNNNAANNNNNCCNNTNNN
DNA Sequence with 11N from front and the first 5N in a row from the back removed
TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
Human genomic plus transcript (Human G+T)Blast Result
Week 1 Protocol Submitting DNA to DNA Sequencing Facility
In this protocal a sample of pgbr22 was send to the DNA sequencing facility for analysis. The sample only contained the DNA template and the sequencing facility added the M13F primer. A nanodrop was conducted to determine the concentration of DNA in the sample.
Virtual gel ladder PCR Primer Design for Primer Overlap Assembly PCR
In this protocal the amino acid sequence for the target proten was taken from the NCBI. Then it was optimize for E. coli class II using http://helixweb.nih.gov/dnaworks/. In the final step we placed the primer already optimize for E. coli class II in the PrimerPlateOrder spreadsheet to have it ordered.
weeks 11 &12
week 12
Protein expression protocol for surrogate target
Docking of NIH_ClinCol3Ded
Protein Characterization special protocol
The band of the protein size was in the nonsoluble fraction due to this it was determined that the protein is insoluble. The change to a surrogate target was made.
week 11
Docking of HF9platesplates
A slight hit was found in this library since the scores were higher than the top ranking ligand in the control docking
Docking of CB_306
The gold results from this docking did not show any good hits. This since the scores were lower than the scores from the control docking
Protocol Virtual screen on protein target
The results of the control docking are shown above this results prove that the control docking on the homology model was a success. The top ranking ligand in this docking was the one found in the protein used for the homology model template.
Protocol Ligand Prep
- Purpose is to prepare the control ligands for virtual screening
Protocol Protein CharacterizationThe ligands were concatenated into a single file and taken through a ligand preparation processes using ICM which prepares the ligands by applying the proper protonation states for pH 7, assigning 3-D coordinates and were energy minimized using the MMFF (Merck Molecular Force Field) prior to doing a control docking.
Protocol Protein Purification
weeks 9&10
Week 10
Protocol Protein Expression Scaled Up
Purpose is to make protein for enzyme assays
- Plate of BL21 cells with inserted vector pNIC-Bsa4 containing the target DNA sequence plates of 10ul
- plate of BL21 cells with inserted vector pNIC-Bsa4 containg the target DNA sequence plate with 50uL coulteure
- option B for the small culture was chosen on friday night the small culture was left to grow overnight
- For this option a small scale growth of bacteria is grown overnight this bacteria will serve as the starting point for the large scale induction step
- For the second day the induction step option B was chosen which is let in shaker incubator for 4 hrs at 37oC
- Bacteria from the Overnight culture was transferred into pre-warm LB media until a 0.1 absorbance at 600nm was obtained at which the LB with the BL21 bacteria was placed in the shaker incubator until a .5 absorbance was obtained at which the IPTG (500 uM final) was added to induce the bacteria into producing the target protein.
- After 4hrs the bacteria was transferred into two 500ml centrifuge containers and spin down at 6000g for 20min. Following this the liquid was discarded and bleached for 20min with 10% bleach. The pellets were resuspended in 12ml of resuspension buffer and they were then transferred into a 15ml conical tube and placed in the -20oC freezer
Protocol Homology ModelPurpose is to find a protein with similar properties as the target protein due to the fact that there is no crystal structure available of the target protein
- This protocol has been delayed due to ICM not working properly duing the week but the homology protein has been obtained and is 2OZ5.
- 20Z5 is the Crystal structure of Mycobacterium tuberculosis protein tyrosine phosphatase PtpB in complex with the specific inhibitor OMTS
Week 9Protocol Ligand Prep
Purpose to obtain and prep positive and negative controls for virtual drug screening of target protein
Data table with properties and specific compounds for negative and positive control ligands this were obtained using the specifications for the protein the negative were obtained at random using compounds with similar characteristics as the postivie controls
Transformation of plasmid in BL21(DE3) cells
Purpose is to insert the plasmid pNIC-Bsa4 with the gene of interest into BL21 expression cells
DNA sequencing analysis
Purpose to blast the sequences obtained from DNA sequencing facility against the known gene of interest in order to confirm a positive clone
Sample 4 Forward primer analysis
Query 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 78 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 137
Query 61 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 138 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 197
Query 121 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 198 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 257
Query 181 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 258 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 317
Query 241 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 318 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 377
Query 301 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 378 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 437
Query 361 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 438 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 497
Query 421 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 498 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 557
Query 481 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 558 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 617
Query 541 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 618 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 677
Query 601 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 678 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 737
Query 661 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 738 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 797
Query 721 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 798 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 857
Query 781 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 840
|||||||||| ||||||||||||||||||||| ||||||||||||||| | || ||||
Sbjct 858 AACGCGGCGTNNGACGAAATCAACGCAAAATACNGTTCTATGGACAACTNCNTCNNGGAG 917
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 897
| |||||||||||||||||| |||||||| |||||||||||||||||||||||||||
Sbjct 918 ANACTGGGTCTGACGGACGCNAAAAAAGANCAGCTGAAGAAAGCGTACCTGTACTAA 974
Sample 1 Forward primer analysis
Query 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 90 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 149
Query 61 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 150 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 209
Query 121 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 210 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 269
Query 181 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 240
||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||
Sbjct 270 AAGACTACCGACGGTCTCACGATCAAACCGC-TAAACTGATCCGTTCTGCGGAACTGGCG 328
Query 241 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 329 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 388
Query 301 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 389 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 448
Query 361 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 449 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 508
Query 421 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 509 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 568
Query 481 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 540
||||| | ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 569 ATCAC-G-TGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 626
Query 541 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 627 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 686
Query 601 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 687 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 746
Query 661 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 747 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 806
Query 721 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 807 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 866
Query 781 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 840
|||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||
Sbjct 867 AACGCGGCGTTCGACGAANTCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 926
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 897
|||||||||||||||||||||||||| |||||||||| ||||||||||||||||||
Sbjct 927 AAACTGGGTCTGACGGACGCGAAAAAN-AACAGCTGAANAAAGCGTACCTGTACTAA 982
Sample 2 Foward sample analysis
Query 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 89 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 148
Query 61 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 149 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 208
Query 121 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 209 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 268
Query 181 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 269 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 328
Query 241 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 329 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 388
Query 301 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 389 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 448
Query 361 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 449 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 508
Query 421 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 509 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 568
Query 481 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 569 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 628
Query 541 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 629 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 688
Query 601 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 689 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 748
Query 661 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 720
|||||||||||||||||||||| ||||||||||||||||||||||||||||||||
Sbjct 749 TCTAACAAATACCGTGCGGACG------AAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 802
Query 721 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 780
|||||||||||||||||||| | ||||||||||||||||||||||||||||||||||||
Sbjct 803 GACAACAAGAAAGTTATCGANNGNATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 862
Query 781 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 840
|||||||||||||||||||||||||| |||||||||||| ||||||||||||||||||||
Sbjct 863 AACGCGGCGTTCGACGAAATCAACGC-AAATACGGTTCTNTGGACAACTTCCTCAAGGAG 921
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 897
|||||||||||||||||||| ||||| | |||||| ||||||||||||||||||
Sbjct 922 AAACTGGGTCTGACGGACGCNAAAAA-GNN-AGCTGAN-AAAGCGTACCTGTACTAA 975
Sample 2 Reverse sample anaylsis
Query 1 ATGAAG-AATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGG 59
|||||| ||| |||||||||||||| ||||||||||||| | |||||||||||||||||
Sbjct 979 ATGAAGNAATNGGGTTAAAGTTACCNGTGCAGGTGTTCTNNCNGCGACCCTGCTGCTGGG 920
Query 60 TGGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCT 119
||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||
Sbjct 919 TGGTTGTGGTGCGCAGTNTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCN 860
Query 120 GAAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTA 179
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 859 GAAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTA 800
Query 180 TAAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGC 239
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 799 TAAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGC 740
Query 240 GAATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGT 299
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 739 GAATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGT 680
Query 300 TGACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGA 359
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 679 TGACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGA 620
Query 360 TTACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGAC 419
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 619 TTACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGAC 560
Query 420 CGCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTT 479
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 559 CGCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTT 500
Query 480 TATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAA 539
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 499 TATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAA 440
Query 540 CCAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCAC 599
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 439 CCAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCAC 380
Query 600 CGCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCT 659
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 379 CGCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCT 320
Query 660 GTCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAAC 719
||||||||||||||||||||||||||| |||||||||||||||||||||||||||
Sbjct 319 GTCTAACAAATACCGTGCGGACGAAAA------GGCGATCGAAGCGGTTGCGGCGAAAAC 266
Query 720 CGACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACAT 779
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 265 CGACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACAT 206
Query 780 TAACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGA 839
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 205 TAACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGA 146
Query 840 GAAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 897
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 145 GAAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 88
Sample 3 Forward sample analysis
Query 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 85 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 144
Query 61 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 145 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 204
Query 121 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 205 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 264
Query 181 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 265 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 324
Query 241 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 325 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 384
Query 301 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 385 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 444
Query 361 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 445 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 504
Query 421 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 505 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 564
Query 481 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 565 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 624
Query 541 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 625 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 684
Query 601 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 685 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 744
Query 661 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 720
||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||
Sbjct 745 TCTAACAAATACCGTGCGGACGAAAAC-AAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 803
Query 721 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 780
||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||
Sbjct 804 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAG-TCGTGAATCTTACATT 862
Query 781 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 840
|||||||||||||||||||||||||| ||||||||||||||||||||||| |||||||||
Sbjct 863 AACGCGGCGTTCGACGAAATCAACGC-AAATACGGTTCTATGGACAACTTNCTCAAGGAG 921
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTA 896
||||||||||||| ||||| ||||| || |||||| |||||||||||||||||
Sbjct 922 AAACTGGGTCTGANNGACGCNAAAAA-GAN-AGCTGAN-AAAGCGTACCTGTACTA 974
Sample 3 Reverse sample analysis
Query 4 AAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGTGGT 63
|||||||||||| ||||| | |||||||||||||||||||| |||||||||||||
Sbjct 970 AAGAATTGGGTTNAAGTTNNCNGTGCAGGTGTTCTGTCCGCG-NNNNGCTGCTGGGTGGT 912
Query 64 TGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTG-AACAGACCCTGAA 122
||||||||||||| |||||||||||||||||||||||||||||||| |||||||| ||||
Sbjct 911 TGTGGTGCGCAGTNTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGNAACAGACCNTGAA 852
Query 123 ACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTATAA 182
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 851 ACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTATAA 792
Query 183 GACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCGAA 242
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 791 GACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCGAA 732
Query 243 TCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTTGA 302
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 731 TCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTTGA 672
Query 303 CTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGATTA 362
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 671 CTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGATTA 612
Query 363 CACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACCGC 422
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 611 CACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACCGC 552
Query 423 GTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTTAT 482
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 551 GTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTTAT 492
Query 483 CACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAACCA 542
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 491 CACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAACCA 432
Query 543 AGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACCGC 602
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 431 AGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACCGC 372
Query 603 GCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTGTC 662
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 371 GCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTGTC 312
Query 663 TAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACCGA 722
||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||
Sbjct 311 TAACAAATACCGTGCGGACGAAAACAAAA-GGCGATCGAAGCGGTTGCGGCGAAAACCGA 253
Query 723 CAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATTAA 782
|||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||
Sbjct 252 CAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGT-CGTGAATCTTACATTAA 194
Query 783 CGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAGAA 842
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 193 CGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAGAA 134
Query 843 ACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 897
|||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 133 ACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 79
Sample 5 Foward sample anaylsis
Query 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTG 39
|||||||||||||||||||||||||||||||||||||||
Sbjct 86 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTG 124
Sample 6 Foward sample anaylsis
Query 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 87 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCTGGGT 146
Query 61 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 147 GGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTG 206
Query 121 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 207 AAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATCTCGGCGGTTAT 266
Query 181 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 267 AAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTGCGGAACTGGCG 326
Query 241 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 327 AATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCGTT 386
Query 301 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 387 GACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGAT 446
Query 361 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 447 TACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCCAAGACCTGACC 506
Query 421 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 507 GCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGAACAAATCTTTT 566
Query 481 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 567 ATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAAC 626
Query 541 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 627 CAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTGGTTTCGGCACC 686
Query 601 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 687 GCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACGACTACATGCTG 746
Query 661 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 747 TCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTGCGGCGAAAACC 806
Query 721 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 807 GACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATT 866
Query 781 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 867 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 926
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTA 896
|||||||||||||||||||| ||||| || |||||| |||||||| ||||||||
Sbjct 927 AAACTGGGTCTGACGGACGCNAAAAA-GAN-AGCTGAN-AAAGCGTANCTGTACTA 979
Sample 7 Foward sample anaylsis
uery 1 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCG-ACC 48
||||||||||||||||||||||||||||||||||||||||||||| |||
Sbjct 85 ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGTACC 133
Sample 6 Reverse sample anaylsis
Query 50 TGCTGCTGGGTGGTTGTGGTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTG 109
||||||||||||| |||| |||||||||||||||||||||||||||||||||||||||||
Sbjct 937 TGCTGCTGGGTGGNTGTGNTGCGCAGTCTGAGGAAAAAGCGGAAGCGAACGTTAAAACTG 878
Query 110 AACAGACCCTGAAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATC 169
|||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 877 AACAGACCNTGAAACCGGGTTCTCAAATCAAACTGGAAGGTGCGGTTAACGTGCGCGATC 818
Query 170 TCGGCGGTTATAAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTG 229
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 817 TCGGCGGTTATAAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCCGTTCTG 758
Query 230 CGGAACTGGCGAATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGT 289
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 757 CGGAACTGGCGAATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGT 698
Query 290 CTCACATCGTTGACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGA 349
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 697 CTCACATCGTTGACTTTCGCACCTCTTCTGAAGTTGCGACCAAACCGGACCCAAAGCTGA 638
Query 350 CCGACGTGGATTACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCC 409
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 637 CCGACGTGGATTACACCCACGACTCTGTTATGAAAGACAACGGTACCTCTACCTCCACCC 578
Query 410 AAGACCTGACCGCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGA 469
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 577 AAGACCTGACCGCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATCAATGCGA 518
Query 470 ACAAATCTTTTATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCC 529
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 517 ACAAATCTTTTATCACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCC 458
Query 530 TGCTCGCAAACCAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTG 589
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 457 TGCTCGCAAACCAAGACGGCTCTGTCCTGTGGCACTGCACTGCGGGTAAAGACCGTGCTG 398
Query 590 GTTTCGGCACCGCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACG 649
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 397 GTTTCGGCACCGCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAAACACCGTTATTGACG 338
Query 650 ACTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTG 709
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 337 ACTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGGTTG 278
Query 710 CGGCGAAAACCGACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTG 769
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 277 CGGCGAAAACCGACAACAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTG 218
Query 770 AATCTTACATTAACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACT 829
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 217 AATCTTACATTAACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACT 158
Query 830 TCCTCAAGGAGAAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACC 889
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 157 TCCTCAAGGAGAAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACC 98
Query 890 TGTACTAA 897
||||||||
Sbjct 97 TGTACTAA 90
Portion of sample 6 foward in order to compare it with the reverse
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTA 896
|||||||||||||||||||| ||||| || |||||| |||||||| ||||||||
Sbjct 927 AAACTGGGTCTGACGGACGCNAAAAA-GAN-AGCTGAN-AAAGCGTANCTGTACTA 979
*positive clone
Sample 4 Reverse Conformation of positive clone
Query 395 CCTCTACCTCCACCCAAGACCTGACCGCGTCTCTCGCGAAGATGGATAACCCGGAAACCT 454
||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 582 CCTNTACCTCCACCCAAGACCTGACCGCGTCTCTCGCGAAGATGGATAACCCGGAAACCT 523
Query 455 TCCTGATCAATGCGAACAAATCTTTTATCACCGATGAAACCTCTATCCAGGCGTACAAAG 514
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 522 TCCTGATCAATGCGAACAAATCTTTTATCACCGATGAAACCTCTATCCAGGCGTACAAAG 463
Query 515 ACTTCTTCGACATCCTGCTCGCAAACCAAGACGGCTCTGTCCTGTGGCACTGCACTGCGG 574
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 462 ACTTCTTCGACATCCTGCTCGCAAACCAAGACGGCTCTGTCCTGTGGCACTGCACTGCGG 403
Query 575 GTAAAGACCGTGCTGGTTTCGGCACCGCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAA 634
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 402 GTAAAGACCGTGCTGGTTTCGGCACCGCGCTGGTTCTCTCTGCACTCGGTGTGGACAAAA 343
Query 635 ACACCGTTATTGACGACTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGG 694
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 342 ACACCGTTATTGACGACTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGG 283
Query 695 CGATCGAAGCGGTTGCGGCGAAAACCGACAACAAGAAAGTTATCGACGGTATGACCGCAG 754
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 282 CGATCGAAGCGGTTGCGGCGAAAACCGACAACAAGAAAGTTATCGACGGTATGACCGCAG 223
Query 755 TTATGGAAGTTCGTGAATCTTACATTAACGCGGCGTTCGACGAAATCAACGCAAAATACG 814
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 222 TTATGGAAGTTCGTGAATCTTACATTAACGCGGCGTTCGACGAAATCAACGCAAAATACG 163
Query 815 GTTCTATGGACAACTTCCTCAAGGAGAAACTGGGTCTGACGGACGCGAAAAAAGAACAGC 874
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 162 GTTCTATGGACAACTTCCTCAAGGAGAAACTGGGTCTGACGGACGCGAAAAAAGAACAGC 103
Query 875 TGAAGAAAGCGTACCTGTACTAA 897
|||||||||||||||||||||||
Sbjct 102 TGAAGAAAGCGTACCTGTACTAA 8
Missing parts of sample 4 forward:
Query 781 AACGCGGCGTTCGACGAAATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAG 840
|||||||||| ||||||||||||||||||||| ||||||||||||||| | || ||||
Sbjct 858 AACGCGGCGTNNGACGAAATCAACGCAAAATACNGTTCTATGGACAACTNCNTCNNGGAG 917
Query 841 AAACTGGGTCTGACGGACGCGAAAAAAGAACAGCTGAAGAAAGCGTACCTGTACTAA 897
| |||||||||||||||||| |||||||| |||||||||||||||||||||||||||
Sbjct 918 ANACTGGGTCTGACGGACGCNAAAAAAGANCAGCTGAAGAAAGCGTACCTGTACTAA 974
Great--looks like you've been working a lot in lab. Captions and analysis could be more thorough. -UM
weeks 7&8
Week 8
Protocol Submit to DNA Sequencing**
- The purpose is to submit the DNA to sequencing facility to see if the transformed gene in DH5alpha with the inserted pNIC is correct
Protocol Midi Prep Kit - HiSpeed (Round 3)- Purpose is to extract DNA from transformed bacterial cells
For this protocol the Midi prep protocol found inside the kit was used to extract the DNA from the transformed bacteria.nanodrop showing the concentration of pNIC-Bsa4 after midipreped
This concentration is excellent to work with for cloning it should make cutting pNIC more pleasing
Protocol Miniprep
- Purpose is to extract DNA from transformed bacterial cells from making master plate protocol
- All the consentartions after nano drop were in the range of 77-95ng/uL this were acceptable consentrations and I proceeded to submit to sequencing
Protocol pNIC-Bsa4 cloningLB+Kan+5%suc plate with DH5alpha and pNIC-Bsa4 with the gene of interest inserted (ratio of pNIC to gene used for this plate was 1microliter:9microliter)
LB+Kan+5%suc plate with DH5alpha and pNIC-Bsa4 with the gene of interest inserted (ratio of pNIC to gene used for this plate was 2microliter:4microliter)
NanoDrop number 1 for cut pNIC Bsa4
NanoDrop number 2 for cut pNIC-Bsa4
**excellent results were obtained from cutting pNIC Bsa4 for the second time will proceed to the next step
Results from nano drop 1 of cut pNIC-Bsa4
Results for nano drop number 2 of cut pNIC-Bsa4
1% Agarose gel for the cut pNIC-Bsa4 round 1 the cut was successful but the final concentration was too low
Due to low level of concentration of the cut pNIC-Bsa4 this will not be useful for the next step the procedure must be redone
Week 7
Protocol transformation for pNIC
- After making LB medium I grew more DH5alpha with the vector pNIC-Bsa4 inserted on them. This was done to have extra pNIC incase that the cutting of pNIC does not work.
Protocol LB Medium- purpose to make LB medium to grow bacteria in.
- made more LB medium to have ready incase that the cutting of pNIC Bsa4 is unsucessful this will serve to grow more bacteria in.
Protocol LB Agar Kan + Suc plates- purpose to make LB + kan + suc plates to used to grow transformed bacteria
- The suc is added to test that the Sac B gene was cut out of pNIC properly since there is a gene that makes a toxic with sucrose which kills the bacteria if the gene is still present in the vector
Protocol Midi Prep Kit-HiSpeed (Round 2)- Round two for Midi Prep results for pNIC-Bsa4 the concentration obtained of pNIC in the second round were slightly better than the first round though it will be hard to do a successful cutting of pNIC Bsa4 an attempt will be done and if unsuccessful results more pNIC will be grown and MIDI preped.
I would like to see more formalized captions bro! Good job with the progress overall though! - Michael T.week 5 & 6
Week 6
Protocol Midi Prep Kit - HiSpeed
- Purpose is to extract DNA from transformed bacterial cells
For this protocol the Midi prep protocol found inside the kit was used to extract the DNA from the transformed bacteria.In the first attempt to do the midiprep the final concentration of pNIC-Bsa4 was extremely low to even attempt the next step the procedure will be redone during week 7.
The results from the first nanodrop of the midiprep
The results of the second nanodrop of the midiprep
Due to the low concentration of pNIC the procedure will be redone during the following week.
Transformation of competent cells for plasmid prep of pNIC-Bsa4(Day2-3)
PyMol Refresher
Chain A shown as red chain on the left, Chain B is shown as yellow on the right. The substrate NAP is shown as sticks green and the substrate DU is shown as sticks blue.
Chain A shown as red chain on the right, Chain B is shown as yellow on the left. The substrate NAP is shown as sticks green and the substrate DU is shown as sticks blue. Showing the hydrophobic residues as magenta, ionic residues as orange and polar as pink.
Showing the active site of 3CL9 shown in green. MTX is shown as sticks colored dark green. The polar contacts of MTX to the protein are shown in black. NAP is shown in cyanide.
Alignment of 1U72 and 3CL9 using (RMSD: 1.23). 1U72 is shown as a ribbion, color red. 3CL9 is shown as a ribbon colored green. the active site residues are shown as sticks yellow. MTX is shown as sticks. The polar contacts of MTX to the respective protein are shown in black dashes.
Protein BLAST of residues in the active sites of 1U72 and 3CL9
showing 3HBB as ribbon. With Chain A shown as blue. Chain B shown as Orange. Chain c shown as yellow. Chain D shown as dark pink.
MTX shown as sticks red, active site of 1U72 shown as yellow sticks, polar contacts shown as black dotted line, 3HBB as ribbon. With Chain A shown as blue. Chain B shown as Orange. Chain c shown as yellow. Chain D shown as dark pink.
This lab helped in remembering the basics of PyMol. This lab served as a good review for the program. Specially since the lab used many of the learned techniques from past Pymol experiments. A lot can be obtain from reviewing this information and specially once we start with the virtual screening process and we need to use the pymol to obtain a visual of whats happening in the docking.
Forward: This lab will help in the analysis of the gold results once we do the virtual screening.
Week 5
Transformation of competent cells for plasmid prep of pNIC-Bsa4(Day1)
- Purpose to transform bacterial plasmid DNA to make more plasmid DNA
In the first day of this process the DH5Alpha was obtained from the -80C fridge and was added to a conical tube then the vector was added and a 30min ice period was waited. The a 42C 45sec heat shock followed. Then a two minute wait on ice and then the SOC media was added. The tube was placed in the shaker incubater for 30min. The last step was to plate the end product and place in the incubator for overnight incubation.The first time doing this procedure the vector was mistakenly not added to the bacteria making it impossible for them to grow in the LB +kan plate due to the fact that the vector contained the Kan resistance gene.
The procedure was done for a second time and the same result happened it was not until the end of the second incubation period that the problem was figured out but already 2 days of work had been lost due to protocol mistake.
On the third try the plates had a great colony growth
The procedure was done correctly and the process was taken into day two for overnight growth in the LB media + kan.
The resulting plate from the overnight growth.
The second plate with the DH5Alpha +kan +LB this plate was where the colony for the overnight cultivation was taken from since the colonies were more clearly seen.
LB Media and Plates Preparation protocol
- Purpose to make LB media and plates to grow DH5Alpha with pNIC-Bsa4 vector.
For the LB media Bacto-tryptone, Bacto-yeast extract, and NaCL were added at the required concentrations specified in the protocol.For the LB Plates the same concentrations were added just that at the end Bacto-agar was added which is what solidifies the mixture in order to be able to plate. In addition at the end before plating Kanamycin was added to avoid other bacteria to grow.
Both the plates and the LB media had to be Autoclaved prior to the use of it in the growth of bacteria. Extreme clean technique was implemented while plating the Plates due to the fact that a completely sterile plate wanted to be obtained in order to have no contamination.
The plates were stored in the 4 degree C fridge and the LB media was stored in the LB media fridge.
PCR Clean up
For the PCR squared clean up I followed the protocol given by Sigma PCR cleanup kit(Red box kit)
The nanodrop results from the first PCR clean up
In the first attempt to do the clean up the final concentration of PCR product was extremely low due to the fact that a user error was made and the PCR reaction was separated into two containers.
The nanodrop for the second PCR squared cleanup
For the second PCR squared clean up the concentration was still way too low for the target concentration. For this PCR Squared clean up it might have been a faulty PCR squared due to the fact that the gel was made but the strength of the bands was not that high.
The nanodrop of the third PCR Squared Cleanup
In the third PCR squared cleanup the concentration was excellent. This should have been due to the fact that PCR squared was doubled and the amount of elution was half. This made it possible to get a great concentration.
PCR2 squared reaction
- Purpose to make extra PCR product to be able to pass it through PCR clean up protocol.
The pcr squared results from the second PCR squared that was done. I forgot to take a gel image of the second PCR squared and I forgot to do a gel for the first PCR squared. But the second pcr squared was successful showing all the bands in the correct size. The thirds PCR squared had to be doubled since for the first two the clean up of the PCR squared was unsuccessful.The gel shows all the lanes with the PCR squared reaction. This lanes show a size of about 1000bp which is about the size of the gene once the tail primers had been added.
Week 3 & 4
De La O - ok, solid work, but upload your original gel images to Wiki. Use more formalized captions. - Dr. B 092713
Week 4
Protocol Primer Dilution VDS
Protocol PCR PrimerOverlap VDS with Q5 Polymerase
#1 primary and secondary PCR gel
#2 Primary PCR
#2 Secondary PCR using the oligo tails as tails
- Secondary PCR was a success the band was in the correct base pairs size. Though this PCR was done using the tails from the oligos mix since the actual tails had not come in by the time I did Secondary PCR. Though from this we can conclude that the Oligos were made correctly since the gene is now at full size.
#3 Secondary PCR using the tails from tail designWeek 3
PCR Primer Design Tails for pNIC-Bsa4 cloning
GC Content 33.3_%
0 mM Mg2+ Tm 57.1_ oC 1.5 mM Mg2+ Tm _65.1 oC 2 mM Mg2+ Tm _67.4 oC
4 mM Mg2+ Tm 67_ oC 6 mM Mg2+ Tm 67.5_ oC
5’ _ TATCCACCTTTACTGTTAGTACAGGTACGC 3’ _ bp
GC Content _43.3%
0 mM Mg2+ Tm 60.4_ oC 1.5 mM Mg2+ Tm 68_ oC 2 mM Mg2+ Tm _67.1 oC
4 mM Mg2+ Tm _69.6 oC 6 mM Mg2+ Tm _70.1__ oC
Primer Dilutions for Assembly Step
Restriction Enzyme Digest
PCR protocol for pGBR22 attempts 2-4
Week 1 & 2
De La O - good job. Take another try at PCR this week. Dr. B 090913
PCR protocol for pGBR22
Analyzing DNA Sequence
- The goal was to determine the DNA sequence found in the plasmid using the DNA sequencing obtain from the DNA sequencing facility. The DNA sequence was also blasted to compare it to a database of human G+T and a second blast was conducted to compare it to a Nucleotide collection (nr/nt). A virual gel ladder was created to determine where would the DNA sequence be found in a gel.
- Original DNA Sequence
- Human genomic plus transcript (Human G+T)Blast Result
Week 1NNNNNNNNNNNTATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANCNNNNNCNTTCNNTTNTNCNNNTNNNNNNNNNNNNNNNTTNCCGNNAGNTNNAANNGGGGNNNNNNNNNATNNGNTNNNNNNNNNNCCAAANTGNNNGNNNNGNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNGANCNNNNNNNNNNNNANNNNNNNNNNNCNNNNNAANNNNNCCNNTNNN
DNA Sequence with 11N from front and the first 5N in a row from the back removed
TATNGNATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCTTCCCACAGTTGCGCAGCCTGATGGCNATGGACGCNNCCTGNANCGNCGCATTAGCNCGGCNGGNNNGNGNNCNCGCAGCGNGACNCTACANTTNCNGCNNCNANC
Protocol Submitting DNA to DNA Sequencing Facility
Virtual gel ladder
PCR Primer Design for Primer Overlap Assembly PCR
- In this protocal the amino acid sequence for the target proten was taken from the NCBI. Then it was optimize for E. coli class II using http://helixweb.nih.gov/dnaworks/. In the final step we placed the primer already optimize for E. coli class II in the PrimerPlateOrder spreadsheet to have it ordered.
- || Lmon_PTP_1 || ATGAAGAATTGGGTTAAAGTTACCGGTGCAGGTGTTCTGTCCGCGACCCTGCTGCT ||
Primer Sequence Ordered for Listeria monocytogenes|| Lmon_PTP_2 || ACGTTCGCTTCCGCTTTTTCCTCAGACTGCGCACCACAACCACCCAGCAGCAGGGTCGCG ||
|| Lmon_PTP_3 || AAAAGCGGAAGCGAACGTTAAAACTGAACAGACCCTGAAACCGGGTTCTCAAATCAAACT ||
|| Lmon_PTP_4 || TTATAACCGCCGAGATCGCGCACGTTAACCGCACCTTCCAGTTTGATTTGAGAACCCGGT ||
|| Lmon_PTP_5 || GCGATCTCGGCGGTTATAAGACTACCGACGGTCTCACGATCAAACCGCATAAACTGATCC ||
|| Lmon_PTP_6 || TCTTATCAGAGTCAGACAGATTCGCCAGTTCCGCAGAACGGATCAGTTTATGCGGTTTGA ||
|| Lmon_PTP_7 || CGAATCTGTCTGACTCTGATAAGAAAAAACTGGTTAACACCTACGACCTGTCTCACATCG ||
|| Lmon_PTP_8 || TCCGGTTTGGTCGCAACTTCAGAAGAGGTGCGAAAGTCAACGATGTGAGACAGGTCGTAG ||
|| Lmon_PTP_9 || GTTGCGACCAAACCGGACCCAAAGCTGACCGACGTGGATTACACCCACGACTCTGTTATG ||
|| Lmon_PTP_10 || GGTCAGGTCTTGGGTGGAGGTAGAGGTACCGTTGTCTTTCATAACAGAGTCGTGGGTGTA ||
|| Lmon_PTP_11 || TCCACCCAAGACCTGACCGCGTCTCTCGCGAAGATGGATAACCCGGAAACCTTCCTGATC ||
|| Lmon_PTP_12 || TGGATAGAGGTTTCATCGGTGATAAAAGATTTGTTCGCATTGATCAGGAAGGTTTCCGGG ||
|| Lmon_PTP_13 || CACCGATGAAACCTCTATCCAGGCGTACAAAGACTTCTTCGACATCCTGCTCGCAAACCA ||
|| Lmon_PTP_14 || CGGTCTTTACCCGCAGTGCAGTGCCACAGGACAGAGCCGTCTTGGTTTGCGAGCAGGATG ||
|| Lmon_PTP_15 || CACTGCGGGTAAAGACCGTGCTGGTTTCGGCACCGCGCTGGTTCTCTCTGCACTCGGTGT ||
|| Lmon_PTP_16 || TGTTAGACAGCATGTAGTCGTCAATAACGGTGTTTTTGTCCACACCGAGTGCAGAGAGAA ||
|| Lmon_PTP_17 || ACGACTACATGCTGTCTAACAAATACCGTGCGGACGAAAACAAAAAGGCGATCGAAGCGG ||
|| Lmon_PTP_18 || GTCATACCGTCGATAACTTTCTTGTTGTCGGTTTTCGCCGCAACCGCTTCGATCGCCTTT ||
|| Lmon_PTP_19 || CAAGAAAGTTATCGACGGTATGACCGCAGTTATGGAAGTTCGTGAATCTTACATTAACGC ||
|| Lmon_PTP_20 || ATAGAACCGTATTTTGCGTTGATTTCGTCGAACGCCGCGTTAATGTAAGATTCACGAACT ||
|| Lmon_PTP_21 || ATCAACGCAAAATACGGTTCTATGGACAACTTCCTCAAGGAGAAACTGGGTCTGACGGAC ||
|| Lmon_PTP_22 || TTAGTACAGGTACGCTTTCTTCAGCTGTTCTTTTTTCGCGTCCGTCAGACCCAGTTTC ||